Generation of Transgene-Free iPSC Lines from Human Normal and Neoplastic Blood Cells Using Episomal Vectors.
ABSTRACT Human induced pluripotent stem cells (iPSCs) have become an important tool for modeling human diseases and are considered a potential source of therapeutic cells. Original methods for iPSC generation use fibroblasts as a cell source for reprogramming and retroviral vectors as a delivery method of the reprogramming factors. However, fibroblasts require extended time for expansion and viral delivery of transgenes results in the integration of vector sequences into the genome which is a source of potential insertion mutagenesis, residual expressions, and reactivation of transgenes during differentiation. Here, we provide a detailed protocol for the efficient generation of transgene-free iPSC lines from human bone marrow and cord blood cells with a single transfection of non-integrating episomal plasmids. This method uses mononuclear bone marrow and cord blood cells, and makes it possible to generate transgene-free iPSCs 1-3 weeks faster than previous methods of reprogramming with fibroblasts. Additionally, we show that this approach can be used for efficient reprogramming of chronic myeloid leukemia cells.
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ABSTRACT: Vision is the sense that we use to navigate the world around us. Thus it is not surprising that blindness is one of people’s most feared maladies. Heritable diseases of the retina, such as age-related macular degeneration and retinitis pigmentosa, are the leading cause of blindness in the developed world, collectively affecting as many as one-third of all people over the age of 75, to some degree. For decades, scientists have dreamed of preventing vision loss or of restoring the vision of patients affected with retinal degeneration through drug therapy, gene augmentation or a cell-based transplantation approach. In this review we will discuss the use of the induced pluripotent stem cell technology to model and develop various treatment modalities for the treatment of inherited retinal degenerative disease. We will focus on the use of iPSCs for interrogation of disease pathophysiology, analysis of drug and gene therapeutics and as a source of autologous cells for cell transplantation and replacement.Progress in Retinal and Eye Research 11/2014; · 9.90 Impact Factor
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ABSTRACT: During stem cell differentiation, various cellular responses occur that are mediated by transcription factors and proteins. This study evaluated the abilities of SOX9, a crucial protein during the early stage of chondrogenesis, and siRNA targeting Cbfa-1, a transcription factor that promotes osteogenesis, to stimulate chondrogenesis. Non-toxic poly-(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) were coated with Cbfa-1-targeting siRNA and loaded with SOX9 protein. Coomassie blue staining and circular dichroism revealed that the loaded SOX9 protein maintained its stability and bioactivity. These NPs easily entered human mesenchymal stem cells (hMSCs) in vitro and caused them to differentiate into chondrocytes. Markers that are typically expressed in mature chondrocytes were examined. These markers were highly expressed at the mRNA and protein levels in hMSCs treated with PLGA NPs coated with Cbfa-1-targeting siRNA and loaded with SOX9 protein. By contrast, these cells did not express osteogenesis-related markers. hMSCs were injected into mice following internalization of PLGA NPs coated with Cbfa-1-targeting siRNA and loaded with SOX9 protein. When the injection site was excised, markers of chondrogenesis were found to be highly expressed at the mRNA and protein levels, similar to the in vitro results. When hMSCs internalized these NPs and were then cultured in vitro or injected into mice, chondrogenesis-related extracellular matrix components were highly expressed.Biomaterials 06/2014; · 8.31 Impact Factor
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ABSTRACT: Human pluripotent stem cells (hPSCs) are increasingly gaining attention in biomedicine as valuable resources to establish patient-derived cell culture models of the cell type known to express the primary pathology. The idea of " a patient in a dish " aims at basic, but also clinical, applications with the promise to mimic individual genetic and metabolic complexities barely reflected in current invertebrate or vertebrate animal model systems. This may particularly be true for the inherited and complex diseases of the retina, as this tissue has anatomical and physiological aspects unique to the human eye. For example, the complex age-related macular degeneration (AMD), the leading cause of blindness in Western societies, can be attributed to a large number of genetic and individual factors with so far unclear modes of mutual interaction. Here, we review the current status and future prospects of utilizing hPSCs, specifically induced pluripotent stem cells (iPSCs), in basic and clinical AMD research, but also in assessing potential treatment options. We provide an outline of concepts for disease modelling and summarize ongoing and projected clinical trials for stem cell-based therapy in late-stage AMD.Journal of Clinical Medicine Research 02/2015; 4(2015 4):282-303.
Uma Lakshmipathy and Mohan C. Vemuri (eds.), Pluripotent Stem Cells: Methods and Protocols, Methods in Molecular Biology,
vol. 997, DOI 10.1007/978-1-62703-348-0_13, © Springer Science+Business Media New York 2013
Generation of Transgene-Free iPSC Lines from Human
Normal and Neoplastic Blood Cells Using Episomal Vectors
Kejin Hu and Igor Slukvin
Human induced pluripotent stem cells (iPSCs) have become an important tool for modeling human
diseases and are considered a potential source of therapeutic cells. Original methods for iPSC generation
use fi broblasts as a cell source for reprogramming and retroviral vectors as a delivery method of the repro-
gramming factors. However, fi broblasts require extended time for expansion and viral delivery of trans-
genes results in the integration of vector sequences into the genome which is a source of potential
insertion mutagenesis, residual expressions, and reactivation of transgenes during differentiation. Here,
we provide a detailed protocol for the ef fi cient generation of transgene-free iPSC lines from human bone
marrow and cord blood cells with a single transfection of non-integrating episomal plasmids. This method
uses mononuclear bone marrow and cord blood cells, and makes it possible to generate transgene-free
iPSCs 1–3 weeks faster than previous methods of reprogramming with fi broblasts. Additionally, we show
that this approach can be used for ef fi cient reprogramming of chronic myeloid leukemia cells.
Key words Epstein–Barr virus , Episomal plasmids , Reprogramming , Induced pluripotent stem cells ,
Human bone marrow , Cord blood , Chronic myeloid leukemia
The iPSC technology offers a novel opportunity for basic research,
disease modeling, drug screening, and cell therapies. Conventional
reprogramming methods rely on gamma retroviral or lentiviral
vectors for the delivery of the reprogramming factors ( 1– 3 ) . Virus-
based techniques cause insertion mutagenesis, residual expression,
and reactivation of transgenes and are of low ef fi ciency. Many
approaches have been investigated to avoid or eliminate integra-
tion of transgenes in the reprogrammed cells including non-inte-
grating adenoviral vectors ( 4 ) , Sendai RNA viral vectors ( 5, 6 ) ,
repeated transient transfection ( 7 ) , protein transduction ( 8, 9 ) ,
RNA transfection ( 10 ) , Cre-LoxP excision system ( 11 ) , PiggyBac
transposon system ( 12, 13 ) , and small molecules ( 14– 20 ) . However,
these alternative approaches still have their limitations such as low
ef fi ciency, multiple rounds of transfection, additional expertise for
164Kejin Hu and Igor Slukvin
RNA and protein preparations, instability of RNA and protein
samples, extra steps for excision of pre-integrated sequences and
subsequent screening, and incomplete removal of the exogenous
EBV-based plasmids exist in mammalian cells as an extrachro-
mosomal entity. EBV-plasmids require only two viral elements for
maintenance in the cells: (1) a short cis sequence which is the latent
origin of plasmid replication ( oriP ), and (2) a transelement of
EBNA1 (Epstein-Barr Nuclear Antigen 1) ( 21 ) . Because up to 5%
of the cells lose EBV plasmids during each cell division ( 22, 23 ) ,
transgene-free cells can be obtained simply by passaging and sub-
cloning ( 24, 25 ) . For these reasons, many laboratories, including
our own, favor the EBV-based episomal vector system for the gen-
eration of iPSCs free of foreign sequences ( 24– 31 ) . For the estab-
lishment of patient-speci fi c iPSC line, starting cells are critical due to
the varied accessibility and reprogrammability of cells. Although
fi broblasts have been traditionally used as a source of cells for
reprogramming, the ef fi ciency of fi broblast reprogramming, espe-
cially adult fi broblasts, is relatively low. Moreover, fi broblasts
require 4–6 weeks for isolation and expansion ( 32, 33 ) . In con-
trast, mature blood cells and their progenitors are the most acces-
sible sources of cells in our body.
We demonstrate that transgene-free human iPSCs can be
obtained from human bone marrow, human cord blood, or puri fi ed
human CD34 + cells using the non-integrating episomal system
( 25 ) . This protocol can reprogram blood cells previously frozen in
liquid nitrogen for over 6 years. While many non-integrating pro-
tocols require multiple rounds of transfections, our protocol uses
only one transfection and does not require puri fi cation of particu-
lar blood subset although the puri fi cation of CD34 + progenitors
can help reprogram cells with higher ef fi ciency. Unlike other pro-
tocols for blood reprogramming that predominantly reprogram T
cell populations ( 5, 34– 36 ) , the population reprogrammed with
our method is neither T nor B cells and thus the reprogrammed
genome is free of recombined genomic DNA that results from
gene rearrangements following maturation of T or B cells.
Additionally, we demonstrate that the episomal vector-based
approach can be used to generate iPSCs from neoplastic bone mar-
row cells from patients with chronic myeloid leukemia (CML) to
model leukemia development in vitro ( 25 ) . The iPSC-based model
provides numerous advantages for the study of neoplastic blood
diseases. It can be used to examine leukemia stem cell potentials at
various stages of differentiation for which it may be dif fi cult to
obtain samples from patients, for example, at the hemangioblast
stage. It also provides a unique opportunity to explore the role of
epigenetic changes in the activation of the oncogene-induced aber-
rant regulatory circuits and to identify cell subsets with distinct
tumor-initiating potential and drug sensitivity.
165Generation of Transgene-Free iPSC Lines from Human Normal…
In this chapter, we describe a detailed protocol for the repro-
gramming of archived normal and neoplastic blood cells with epi-
somal constructs. Figure 1 outlines the major steps of the
reprogramming protocol. The procedure starts with a short expan-
sion of the mononuclear cells in the serum-free hematopoietic expan-
sion media followed by nucleofection, an additional expansion for
2 days in the hematopoietic media and a transfer of cells onto mouse
embryonic fi broblast (MEF) feeders. Reprogramming proceeds on
MEF feeders in the standard iPSC medium for 10 days. Additional
culture in MEF-conditioned media for another 10 days is required to
grow typical iPSC colonies which can be handpicked for expansion
and the fi nal establishment of iPSC lines. Although we initially devel-
oped this method for reprogramming mononuclear cells from bone
marrow and cord blood ( 25 ) , we found that the same protocol works
well for reprogramming of CD34 + cells isolated from these sources.
All reagents should be cell-culture grade. Aseptic practice should
be observed for all steps.
1. Recombinant human IL-3 (PeproTech).
2. Recombinant human IL-6 (PeproTech).
3. Recombinant human SCF (PeproTech).
4. Recombinant human Flt3L (PeproTech).
5. Recombinant human FGF-basic (PeproTech).
6. Zebra fi sh FGF-basic (gift from James Thomson, made in house).
7. StemSpan SFEM (Serum-Free Medium for Expansion of
Hematopoietic cells) (Stemcell Technologies).
8. Histopaque-1077 ® (Sigma-Aldrich).
9. Percoll ® (Sigma-Aldrich).
( See Note 1 )
Begin using MEF-CM
on day 10-12
iPSC media, change every
the other day
Conditioned iPSC media
Pick up iPSC colony
on day 17-21
Expansion media with cytokines
Fig. 1 Summary of protocols for reprogramming blood cells using episomal plasmids. Arrows indicate time
points at which the indicated steps are carried out. Broken arrows designate the optional steps. O/N means
overnight recovery by culturing of frozen whole bone marrow or whole cord blood overnight in hematopoietic
expansion medium and fractioning for mononuclear cells with Histopaque gradient centrifugation. CM is
166Kejin Hu and Igor Slukvin
10. EX-CYTE ® growth enhancement media supplement (Celliance).
11. Human cord blood mononuclear cells (AllCells, CA, USA).
12. Human bone marrow mononuclear cells (AllCells, CA, USA).
13. Human bone marrow cells from patient with CML (AllCells,
14. Human cord blood CD34 + cells (AllCells, CA, USA).
15. Human bone marrow CD34 + cells (AllCells, CA, USA).
16. DNase I (Promega).
17. L -Glutamine (Gibco).
18. γ -Irradiated MEF (WiCell).
19. Knockout serum replacement for ESC/iPSC (Gibco).
20. β -mercaptoethanol (Sigma).
21. D-MEM/F-12 (Gibco).
22. MEM nonessential amino acids solution (100×, 10 mM,
HyClone ® ).
23. Penicillin-Streptomycin solution (100×, Cellgro ® ).
24. Hyclone ® Fetal Bovine Serum (de fi ned) (Thermo Scienti fi c).
25. Collagenase type IV (Gibco).
26. Dimethyl sulfoxide (DMSO) (Sigma).
27. Gelatin (Sigma).
28. PBS (without calcium, without magnesium) (Hyclone ® ).
29. Sodium bicarbonate (Fisher Scienti fi c).
30. Thiazovivin (Stemgent ® ).
1. Amaxa ® Human CD34 + Cell Nucleofector ® Kit (Lonza).
1. pEP4-EO2S-ET2K (Addgene).
2. pEP4-EO2S-EN2K (Addgene).
3. pCEP4-M2L (Addgene).
1. Nucleofector ® II (Amaxa Biosystem).
The iPSC medium is, in essence, ESC growth medium with a
higher concentration of FGF2. It consists of 20% Knockout serum
replacement (KOSR), 80% D-MEM/F-12, 1 mM L -glutamine,
0.1 mM β -mercaptoethanol, 1× nonessential amino acid (NEAA)
(0.1 mM). We use 100 ng/ml of recombinant zebra fi sh FGF-
basic, or 10 ng/ml of recombinant human FGF2. It can be stored
for up to 2 weeks at 4°C.
2.2 Transfection Kit
( See Note 2 )
2.4 Key Equipment
( See Note 2 )
2.5 iPSC Medium
( See Note 3 )
167Generation of Transgene-Free iPSC Lines from Human Normal…
The hematopoietic expansion medium consists of StemSpan SFEM
(serum-free expansion media) supplemented with 0.2% Ex-Cyte
(Celliance), recombinant human IL-3 (10 ng/ml), recombinant
human IL-6 (100 ng/ml), recombinant human SCF (100 ng/ml),
and FMS-related tyrosine kinase-3 ligand (Flt-3 L, 100 ng/ml),
100 IU of penicillin, and 100 μ g/ml of streptomycin. Upon arrival,
StemSpan SFEM is aliquoted into 50-ml tubes, and stored at
−20°C or −80°C. Cytokine, growth factors, lipid supplement, and
antibiotics are added immediately before use.
For the preparation of MEF-conditioned medium, we use the
modi fi ed method of Xu et al. ( 37 ) . Add 15 ml of iPSC medium
composed of 10% KOSR, 90% D-MEM/F-12, 0.5 mM L-glutamine,
0.5× NEAA, without FGF2 to a 10-cm tissue dish preseeded with
MEF at a density of 2 × 10 4 /cm 2 . After 24 h of conditioning,
medium is collected. The same dish of MEF can be used for up to
10 times. The collected conditioned media can be stored at −20°C
for over 1 month. To make 500 ml of a working MEF-conditioned
medium for reprogramming, combine the following: 450 ml of
conditioned medium, 50 ml of fresh KOSR, 2.5 ml of 100 mM L -glu-
tamine, 2.5 ml of 100× NEAA, 2 μ l of β -mercaptoethanol, and
appropriate amount of FGF to make a fi nal concentration of
100 ng/ml for zebra fi sh FGF, or 10 ng/ml for recombinant
human FGF2. The MEF-CM is sterilized by fi ltering through a
0.22- μ m fi lter.
2× iPSC freezing medium is composed of 60% Hyclone ® FBS, 20%
DMSO, and 20% basal iPSC media. Prepare freshly when needed
and keep chilled on ice.
Weigh 1 g of gelatin and put it into a 1-L Pyrex bottle; add 1 L of
pure water. Autoclave to sterilize the solution. The gelatin can be
stored at 4°C for over a year.
The working concentration of collagenase IV is 1 mg/ml in plain
D-MEM/F-12 medium. Weigh 45 mg of collagenase IV; put it
into a 50-ml tube; add 45 ml of D-MEM/F-12 medium into the
50-ml tube containing the collagenase IV; close the tube with a
cap; mix well by shaking; remove the cap; sterilize the collagenase
IV solution by fi ltration with a Steri fl ip ® 50-ml fi lter (0.22 μ m,
Millipore). The solution can be stored for up to 14 days at 4°C.
Coat the 6-well plate or 10-cm dish with 0.1% gelatin (2 ml per
well, and 10 ml per dish) overnight at 37°C. Next day, remove the
plate/dish from the incubator and aspirate the gelatin solution from
the plate/dish. Seed 2.5 ml of irradiated MEF at 0.75 × 10 5 /ml into
each well of a 6-well plate (around 2 × 10 4 /cm 2 ), or 15 ml of the
irradiated MEF (0.75 × 10 5 cells/ml) into one 10-cm tissue culture
dish. Culture the MEF overnight at 37°C, 5% CO 2 before use.
( See Note 4 )
2.8 2× iPSC Freezing
2.9 0.1% Gelatin
of Collagenase IV
2.11 MEF Preparation
for Culture of Human
( See Note 5 )
168Kejin Hu and Igor Slukvin
5× Percoll solution : Mix 45 ml of Percoll ® (Sigma, p1644) (sterile)
with 5 ml of 10× PBS (sterilized by fi ltration), which results in 90%
Percoll ® in 1×PBS. Store the 5× Percoll stock solution at 4°C.
1× Percoll solution : Mix 10 ml of 5× Percoll ® solution with
40 ml of sterile 1×PBS solution. The fi nal Percoll ® solution is 18%
in 1× PBS. Smaller volume can be prepared similarly. The 1×
Percoll solution can be prepared before use.
1. Take one vial of blood mononuclear cells from liquid nitrogen
tank, and thaw the cells quickly in a 37°C water bath; transfer
the cells into the 15-ml tube containing 10 ml of plain SFEM
medium ( see Note 7 ).
Centrifuge the cells at 400 × g for 8–10 min.
2. Aspirate the supernatant, add 10 ml of fresh plain SFEM to the
cell pellet, and repeat the washing.
3. Resuspend cells in 1 ml of hematopoietic expansion medium
and count the cells with trypan blue. Add appropriate volume of
hematopoietic expansion medium to the cell suspension to make
the fi nal concentration 1–2 millions cells per ml ( see Note 8 ).
4. Put the cells in one well of a 6-well plate if the cell suspension
volume is 4 ml or less; culture the cells in a T-25 tissue fl ask if
cell suspension volume is 5–10 ml.
5. Culture at 37°C and 5% CO 2 for 2 days.
1. On day 2 (48 h of culture in hematopoietic expansion media),
transfer the cell suspension into a 15 ml tube ( see Note 9 ).
2. Underlay cell suspension with 1.0–1.5 ml of Percoll solution.
3. Centrifuge at 300 × g for 20 min at room temperature.
4. During the centrifugation, take out the nucleofection kit from
4°C, and DNA from −80°C. Put 82 μ l of nucleofection buffer
into a sterile 1.5-ml tube and add 18 μ l of the supplement
(both are supplied as a components of Amaxa ® Human CD34 +
Cell Nucleofector ® Kit) into the buffer; mix well. During this
period of time, the buffer can be brought to room temperature
( see Notes 10 and 11 ).
5. After centrifugation, aspirate the supernatant and interface
containing dead cells and debris without disturbing cell pellet;
resuspend the cell pellet with 10 ml of SFEM, and centrifuge
at 400 × g for 8 min ( see Note 12 ).
6. During the washing, turn on the Nucleofector II. Set up the pro-
gram to U-008 (U-08 for Nucleofector ® I). Prepare one Amaxa
cuvette and one Amaxa transfer pipette in the biosafety hood.
of Percoll ® Solution
3.1 Preparation of
Cells for Nucleofection
( See Note 6 )
of Blood Cells and
169Generation of Transgene-Free iPSC Lines from Human Normal…
7. Add the following DNA plasmids into the buffer mixture prepared
in step 4: 9 μ g of pEP4-EO2S-ET2K, 9 μ g of pEP4-EO2S-EN2K,
and 6 μ g of pCEP4-M2L. Mix well by gentle pipetting, being
careful to avoid introducing bubbles. ( See Note 13 ).
8. Carefully aspirate the entire media from cells in step 5 without
disturbing cells ( see Note 14 ).
9. Add buffer containing DNA ( step 7 ) to the 15-ml tube con-
taining the cells to be transfected ( step 8 ); mix well by gentle
pipetting, being careful to avoid introducing bubbles. Carefully
transfer the cells and DNA in buffer into the transfection
cuvette supplied with Amaxa ® Nucleofector ® Kit. Do not intro-
duce any bubbles. Gently but quickly tap the cuvette fi ve to ten
times immediately to remove any bubbles.
10. Nucleofect the blood cells using program U-008 ( see Note 15 )
11. Add 500 μ l of hematopoietic expansion medium into the
cuvette; mix by one gentle pipetting motion, and transfer cells
into dish or wells containing hematopoietic expansion medium
( see Note 16 ).
12. Culture for 2 days in hematopoietic expansion medium at
37°C, 5% CO 2 .
13. On day 2 after transfection, remove dead cells using Percoll ®
separation as described in steps 1 – 3 and 5 . Transfer the cells
onto a 10-cm dish preseeded with MEFs.
14. Change medium every other day for the fi rst 10 days ( see
Note 17 ).
15. On day 10, start to use MEF-conditioned medium and change
media every day.
16. iPSC colonies should appear between days 17 and 21. Under a
microscope in the biosafety hood, scrape off and pick up the
entire single iPSC colony ( see Fig. 2 ) with a P200 tip. Transfer
colony into a sterile 1.5-ml tube and separate cells by pipetting
fi ve times. Put one individual iPSC colony into one well of a
6-well plate. The fi rst culture from this picked colony is consid-
ered a passage 1 iPSCs (P 1 ) ( see Note 19 ).
17. Grow the P1 cells in iPSC medium at 37°C, 5% CO 2 . Change
the medium every day.
18. When new colonies reach the normal size, repeat step 16 if any
incompletely reprogrammed colonies exist, and grow the P 2
iPSCs at 37°C, 5% CO 2 ( see Note 20 ).
19. Between days 5 and 10 after culture, depending on the density
and colony size, passage the new iPSC line using standard pro-
tocol for passaging human ESC/iPSCs on MEFs (now P 3 ).
20. Between days 5 and 7, freeze the newly established lines
( see freezing protocol below). Passage the remaining wells
170Kejin Hu and Igor Slukvin
for characterization and further freezing at higher passages.
Established iPSC lines should be analyzed for expression of
pluripotency markers by fl ow cytometry, immuno fl uorescence,
RT-PCR, or gene pro fi ling. Pluripotent potential of these
cells should be evaluated using functional tests including tera-
toma generation, and in vitro differentiation into different
lineages. The established lines should also be karyotyped to
ensure genomic integrity. Characterization of iPSCs
obtained from patient with chronic myeloid leukemia is
shown in Fig. 3 .
1. Warm up collagenase IV in a 37°C water bath for 10 min.
2. Take out the iPSC plate from the incubator.
3. Use 1.5 ml of collagenase IV solution for one well of iPSCs of
a 6-well plate.
4. Incubate at 37°C for 5 min.
5. Aspirate the collagenase IV solution.
3.3 Freezing iPSCs
Fig. 2 Morphology of reprogrammed cell colonies. Panel ( a ) shows a high-quality colony. The colony is bright
and composed of small, tightly packed cells. Only a few incompletely reprogrammed spindle cells are present
at the periphery of the colony. Panels ( b ) and ( c ) are other examples of well-reprogrammed colonies. Completely
reprogrammed cells within colonies are circled. However, more incompletely reprogrammed spindle-like cells
can be seen at the periphery of colony ( arrow ). Panel ( d ) shows a colony at a less advanced stage of repro-
gramming. However, this colony can be picked up and used to establish iPSC lines after transferring to new
MEFs to facilitate complete transition to pluripotency. Bars, 300 μ m. Images of transfected human bone mar-
row mononuclear cells were taken on day 17 of culture on MEFs (day 19 after transfection)
G G BM
1517 H1Tb K562
Fig. 3 Demonstration of pluripotency of iPSC lines generated from bone marrow of a patient with chronic
myeloid leukemia (CML) in chronic phase. ( a ) Flow cytometric analysis of hESC-speci fi c marker expression in
CMLiPSC15 line (surface staining is shown for ALP, TRA-1-60, SSEA3, SSEA4, and TRA-1-81; intracellular stain-
ing is shown for OCT4, SOX2, and NANOG). ( b ) Typical morphology of CMLiPSC15 colony growing on MEFs. ( c )
H&E staining of teratoma from CMLiPSC15 line. Neural rosettes ( upper panel ), cartilage ( middle panel ), and
intestine-like structure ( lower panel ) are shown. ( d ) PCR analysis of genomic (G) and episomal (E) DNA demon-
strates that CMLiPSCs are transgene-free. Vector (EBNA)- and transgene (T)-speci fi c primers were used as
indicated. RT-PCR analysis of expression of transgenes (T) and endogenous pluripotency genes and BCR-ABL in
CMLiPSC lines 15 and 17. H1 is human embryonic stem cell line H1. BM is bone marrow mononuclear cells.
K562 is myeloid leukemia line established from a patient with chronic myeloid leukemia in blast crisis. Tb is
positive controls, BM cells transfected with the same reprogramming plasmids
172Kejin Hu and Igor Slukvin
6. Add 3 ml of iPSC medium and gently wash off the iPSC colonies
from the MEF feeder with a 5-ml pipette.
7. Put the iPSCs into a 15-ml tube and centrifuge at 200 × g for
8. Aspirate the washing medium.
9. Add 0.5 ml of iPSC media (ice cold), and add 0.5 ml of iPSC
freezing media (ice cold) to cell pellet.
10. Resuspend cells and transfer cells into a cryotube (1.8 ml).
Avoid excessive pipetting during cell transfer.
11. Put the cryotube in the freezing container, and start the freezing
process immediately in −80°C freezer; the following day, trans-
fer the iPSCs into a liquid N 2 storage tank for the long-term
1. All stock solutions of cytokines are prepared at 1,000×,
aliquoted and kept at −80°C.
2. Blood progenitors are dif fi cult to transfect using the traditional
nonviral transfection methods due to their quiescent nature
and suspension growth ( 38 ) . Nucleofection is critical for suc-
cessful reprogramming of blood progenitor cells. Nucleofection
directly delivers the DNA into the nuclei of cells and results in
early and strong expression of transgenes.
3. Recombinant zebra fi sh FGF-basic can be used instead of
human FGF2. However it is less stable at 37°C and must be
used at concentration of 100 ng/ml. For commercial recombi-
nant human FGF2, we recommend a concentration of
4. Standard protocol for preparation of MEF-conditioned media
requires high density of MEF to be plated in T-75 fl ask
(5.4 × 10 4 /cm 2 ). We found that the routine 2 × 10 4 /cm 2 den-
sity of MEF used for ESCs culturing works well for preparation
of MEF-conditioned media and simpli fi es the procedure.
5. The complete MEF preparation protocol can be found in
WiCell protocols: SOP-CC-003B; SOP-CC-006D; SOP-CC-
031D; SOP-CC-021A; SOP-CC-009A; SOP-CC-013A
( https://www.wicell.org ).
6. We grow bone marrow and cord blood mononuclear cells for
2 days before transfection to amplify hematopoietic progeni-
tors. We also recommend a 2-day expansion before transfec-
tion for CD34 + cells isolated using magnetic beads. The
electroporator creates an electromagnetic fi eld in the cell solu-
tion which could affect viability of the puri fi ed CD34 + cells
173Generation of Transgene-Free iPSC Lines from Human Normal…
when magnetic beads attached to them. However, after 2 days
of culture, the beads detach and degrade so that the cells can
be safely electroporated.
7. If unprocessed bone marrow or cord blood is used, a standard
Histopaque gradient centrifugation step should be carried out
to obtain the mononuclear cell fraction. The current protocol
is based on mononuclear cells.
8. For bone marrow mononuclear cells and CD34 + cells, 1–2 × 10 6
is typically suf fi cient for reprogramming. However, for cord
blood mononuclear cells, 5–10 × 10 6 cells are required for
9. Mature blood cells die in hematopoietic expansion medium
and result in cell clumps. If cell clumping occurs, treat the cells
with DNase I at a concentration of 200 units/ml for 30 min
before Percoll ® puri fi cation. DNase I treatment removes the
DNA released by the dead cells and alleviate the clumping.
10. The shelf life of buffer after mixture with supplement is
3 months. We usually mix these two parts of the reaction just
before transfection to ensure high transfection ef fi ciency.
11. The optimal volume of the reaction buffer for the Nucleofector
device is 100 μ l. Signi fi cant changes to the reaction volume
will result in transfection failure.
12. If puri fi ed CD34 + cells are used for reprogramming, Percoll ®
separation is not required due to the low number of dead cells.
13. Concentration of plasmid DNA in stock solution should be
> 1.5 μ g/ μ l to avoid signi fi cant dilution of nucleofection buf-
fer following DNA addition. Excessive dilution of nucleofec-
tion buffer decreases transfection ef fi ciency.
14. Washing medium must be completely removed before adding
nucleofection buffer. This should be done very carefully to
avoid aspiration and loss of cells. Tilt the tube so that the bot-
tom is elevated to let liquid to fl ow down toward the mouth of
the tube. The liquid can be collected by keeping the tip of the
running vacuum pipette immediately downstream of the fl ow.
This method allows for effective liquid removal and avoids dis-
ruption of the cell pellet.
15. Do not keep cells in the transfection buffer longer than 20 min.
Extended contact time with buffer reduces the viability of cells
and the gene transfer ef fi ciency. After adding buffer, proceed
with electroporation as quick as possible.
16. After nucleofection procedure, cells become friable. It is criti-
cal to use a pipette with larger bore size supplied with the
nucleofection kit for gentle handling of cells.
17. Blood cells grow in suspension and transfected cells gradually
become adherent. However, during the fi rst few days following
174Kejin Hu and Igor Slukvin
reprogramming, many cells still grow in suspension. To avoid
loss of cells following medium change, aspirated medium can
be centrifuged to collect fl oating cells and reseeded back onto
the same MEF dish.
18. MEF cultures should be prepared fresh for plating transfected
cells. Because reprogramming requires extended cultures, the
use of 3-day or older MEF plates will compromise the experi-
ment due to the deterioration of the MEFs.
19. Sometimes no typical colonies appear following the fi rst round
of transfected cells on MEFs (P 0 ). Passaging P 0 cells onto new
MEFs may produce high-quality iPSCs colonies. The culture
ratio may vary from 1:3 to 1:6 depending on the density of the
colonies in the P 0 dishes/plates. The reprogramming ef fi ciency
can be signi fi cantly increased by adding thiazovivin (1 μ M) to
reprogramming cultures. Thiazovivin can be added as early as
the transfected cells are transferred onto MEF feeders or after
the fi rst passage.
20. The second picking will eliminate the contaminated incom-
pletely reprogrammed cells from the fi rst culture. Multiple
colonies can be cultured in the second round of culture.
We thank Professor James Thomson at Morgridge Institute for
Research, Madison, WI, for providing reprogramming plasmids
and FGF2, and Patricia Liu for editorial assistance. This work was
supported by funds from the National Institute of Health (P01
GM081629 and P51RR000167).
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